Characterization of Ca2+ATPase Residues Involved in Substrate and Cation Binding
| العنوان: | Characterization of Ca2+ATPase Residues Involved in Substrate and Cation Binding |
|---|---|
| المؤلفون: | Hailun Ma, Giuseppe Inesi, Suming Hua, Chikashi Toyoshima |
| المصدر: | Annals of the New York Academy of Sciences. 986:63-71 |
| بيانات النشر: | Wiley, 2003. |
| سنة النشر: | 2003 |
| مصطلحات موضوعية: | Models, Molecular, Cation binding, Cations, Divalent, Protein Conformation, Stereochemistry, Iron, ATPase, Mutant, Calcium-Transporting ATPases, Transfection, Cleavage (embryo), General Biochemistry, Genetics and Molecular Biology, Substrate Specificity, Enzyme activator, History and Philosophy of Science, Chlorocebus aethiops, Animals, Magnesium, Phosphorylation, Muscle, Skeletal, chemistry.chemical_classification, Binding Sites, biology, General Neuroscience, Cooperative binding, Proteinase K, Sarcoplasmic Reticulum, Enzyme, Amino Acid Substitution, chemistry, Biochemistry, COS Cells, biology.protein, Rabbits, Oxidation-Reduction |
| الوصف: | The role of amino acid residues involved in substrate and cation binding was investigated in complementary experiments on Fe(2+)-catalyzed oxidation and cleavage, limited digestion with proteinase K, and mutational analysis. Cleavage at Ser346 was produced by Fe(2+) in the presence of substrate (ATP or AMP-PNP) and Ca(2+), and was attributed to Fe(2+) bound to a Mg(2+) site near Ser346 and neighboring Glu696. Ca(2+)- and ATP-dependent oxidation of the Thr441 side chain was also observed and attributed to Fe(2+) substituting for Mg(2+) in the Mg(2+)-ATP complex bound to the N domain. Mutation of Arg560 or Glu439 within the N domain interfered with nucleotide-dependent ATPase resistance to digestion with proteinase K. Furthermore, mutation of Lys352, Lys684, Thr353, Asp703, or Asp707 within the P domain produced similar interference, consistent with a role of these residues in substrate stabilization at the catalytic site. In a third group of experiments, equilibrium isotherms were obtained with Asn796Ala and Glu309Gln mutants, demonstrating non-cooperative binding of one Ca(2+) per ATPase, as opposed to cooperative binding of two Ca(2+) by WT enzyme. No high-affinity binding by Asp800Asn, Glu771Gln, and Thr799Ala mutants was detected. It was also demonstrated that the conformational transitions involved in enzyme activation and interconversion of Ca(2+) binding and phosphorylation energy, are triggered by Ca(2+) binding to site II and stabilization of Glu309 (M4) and N796 (M6). |
| تدمد: | 1749-6632 0077-8923 |
| DOI: | 10.1111/j.1749-6632.2003.tb07140.x |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::df5ffd02454ebcfbb125766da0319d88 https://doi.org/10.1111/j.1749-6632.2003.tb07140.x |
| Rights: | CLOSED |
| رقم الانضمام: | edsair.doi.dedup.....df5ffd02454ebcfbb125766da0319d88 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 17496632 00778923 |
|---|---|
| DOI: | 10.1111/j.1749-6632.2003.tb07140.x |